The Role And Mechanism Of LINC00973 In NSCLC

Objective: To screen LncRNAs through high-throughput sequencing,determine the expression change of LINC00973 in NSCLC,investigate its biological function and molecular mechanism,provide a new biomarker for the diagnosis and treatment of NSCLC.Methods:RNA sequencing was performed to identify the differential expression patterns of LncRNAs between NSCLC tissues and adjacent normal tissues.q RT-PCR was used to detected LINC00973 in NSCLC cell lines and tissues.Melting curve analysis and agarose gel electrophoresis were performed to verify the specificity of the q RT-PCR product,ROC curve analysis and clinicopathological data analysis were used to evaluate the value of its diagnosis and prognosis.siRNA against LINC00973 was used to knockdown and LINC00973-overexpressing plasmid was used to overexpress LINC00973 in NSCLC cells.Cell growth curve assay and colony formation assay were conducted to detect the proliferation ability of NSCLC cells.Transwell migration test and matrix gel invasion test were used to detect the metastasis ability of NSCLC cells.Flow cytometry was performed to detect the effect of LINC00973 on cell cycle and apoptosis.Western blot was used to detect the expression change of related proteins.RNA sequencing was performed to obtain the down regulated mRNAs and the inhibited signaling pathway after LINC00973 knockdown.q RT-PCR and Western blot were carried out to verify sequencing results.The proteins binding to LINC00973 were collected by TRAP and identified by liquid chromatograph-mass spectrometry.RIP was used to detect whether LINC00973 could bind to this protein.The effects of overexpression and knockdown of LINC00973 on the expression of binding protein were examined by Western blot.siRNA against DTX3 L was used to knockdown DTX3 L in NSCLC cells.Cell growth curve assay and colony formation assay were conducted to detect the proliferation ability of NSCLC cells.Flow cytometry was performed to detect the effect of DTX3 L on apoptosis.Results: LINC00973 was highly expressed in NSCLC cells and tissues.Knockdown of LINC00973 attenuated the proliferation and metastasis of NSCLC cells,induced cell cycle arrest at G1 phase and promoted apoptosis.LINC00973 silence downregulated the expression of EMT and proliferation related proteins.Knockdown of LINC00973 resulted in the down-regulation of several tumor related genes such as MMP9 and VCAM1,and the inhibition of Akt-m TOR signaling pathway.Overexpression of LINC00973 had the opposite effect.LINC00973 could directly bind to DTX3L/ PARP9 protein complex and was positively correlated with DTX3 L protein expression.Knockdown of DTX3 L attenuated the proliferation and promoted the apoptosis of NSCLC cells.Conclusions: LINC00973 is up-regulated in NSCLC and could be a new diagnostic marker.LINC00973 may promote NSCLC progression by binding to DTX3L/ PARP9 protein complex.